In this work, we demonstrate an on-chip microfluidic flow cytometry system based on a three-dimensional (3D) hydrodynamic focusing technique, microfluidic drifting. By inducing Dean flow in a curved microfluidic channel, microfluidic drifting can be used to hydrodynamically focus cells or particles in the vertical direction and enables the 3D hydrodynamic focusing in a single-layer planar microfluidic device. Through theoretical calculation, numerical simulation, and experimental characterization, we found that the microfluidic drifting technique can be effectively applied to three-dimensionally focus microparticles with density and size equivalent to those of human CD4+ T lymphocytes. In addition, we developed a flow cytometry platform by integrating the 3D focusing device with a laser-induced fluorescence (LIF) detection system. The system was shown to provide effective high-throughput flow cytometry measurements at a rate of greater than 1700 cells s(-1).